CRYPTDIN EFFECTS ON CRYPT EPITHELIA

描述(申请人摘要)：本提案的目标 是为了阐明隐匿蛋白诱导的隐窝通道形成的生物学 上皮细胞。隐蛋白是肠道(潘氏细胞)特有的α-防御素- 阳离子多肽最早被认为是中性粒细胞的产物，显示出强大的 抗菌性能。已经描述了19种加密素亚型 小鼠和两个，HD-5和HD-6，在人类。黄曲霉毒素的抑菌作用 阿尔法-防御素被认为存在于它们将其划分为 生物膜，并产生阴离子导电孔。Cryptdins、HD-5和HD-6 已知从潘氏细胞释放到隐窝腔内，因此其他 隐窝细胞类型，主要是分泌“未分化”的氯化物。 隐窝细胞，自然地暴露于这些通道形成的多肽。圆周率和 他的合作者最近发现，加密蛋白2和3可以诱导 人肠道T84细胞生理性氯离子分泌反应可能通过 在根尖(腔)膜上形成阴离子传导通道。研究 提出的将检验Paneth细胞衍生的加密蛋白作用于 旁分泌插入法调节肠腺的生理功能 (自组装)氯化物传导孔进入根尖膜 邻近的隐窝上皮细胞与内吞作用和 根尖膜修复。体外实验将通过以下方式定义这一机制 哪些小鼠隐含蛋白2和3可诱导氯的分泌。私家侦探和他的 合作者将使用Matrilysin-/-小鼠和CR2-tox176小鼠来阐明 隐球蛋白对体内外小肠功能的影响。这个 加密素3、HD-5和HD-6的生物物理性质和结构功能 将在完整和半透性的T84和肾脏HEK细胞模型中进行检测 系统和单通道膜片钳。最后，建议进行以下研究： 阐明密码蛋白3诱导通道形成的细胞机制(S) 下调监管。这些研究的重要性通过以下方式得到强调 越来越多的证据表明肠道隐窝在 肠上皮细胞个体发育、生理学(溶质和水运输)、 特异性和非特异性黏膜防御。隐含蛋白在旁分泌中起作用 调节隐窝细胞功能的时尚，似乎有一种独特的机制 行动的一部分。拟议的研究将阐明这些小说的生物学 α-防御素，可能为未来α-防御素的发展奠定基础 抑制剂或使用这些(或相关的多肽)来治疗疾病 肠道或呼吸道的粘膜表面。

DESCRIPTION ( applicant's abstract): The goal of this proposal is to elucidate the biology of crytpdin-induced channel formation in crypt epithelia. Cryptdins are intestinal (Paneth cell) specific alpha-defensins - cationic peptides first recognized as neutrophil products, which exhibit potent antimicrobial properties. Nineteen cryptdin isoforms have been described in the mouse and two, HD-5 and HD-6, in the human. The antimicrobial action of alpha-defensins is thought to reside in their ability to partition into biomembranes and produce anion conductive pores. Cryptdins, and HD-5 and HD-6 are known to be released from Paneth cells into the crypt lumen and thus other crypt cell types, the major one being the chloride secreting "undifferentiated" crypt cell, are naturally exposed to these channel forming peptides. The PI and his collaborators have recently shown the cryptdins 2 and 3 induce a physiologic chloride secretory response in human intestinal T84 cells likely by forming anion conductive channels in the apical (lumenal) membrane. Studies proposed will test the hypothesis that Paneth cell-derived cryptdins act to regulate the physiology of the intestinal crypt by paracrine insertion (self-assembly) of chloride conducting pores into apical membranes of neighboring crypt epithelial cells and down regulation by endocytosis and apical membrane restitution. In vitro experiments will define the mechanism by which mouse cryptdins 2 and 3 induce chloride secretion. The PI and his collaborators will use matrilysin -/- mouse and CR2-tox176 mouse to elucidate the effects of cryptdins on small intestinal functions ex vivo and in vivo. The biophysical properties and structure function of cryptdin 3 and HD-5 and HD-6 will be examined in intact and semi-permeabilized T84 and renal HEK cell model systems and by single channel patch clamp. Finally studies are proposed to elucidate the cellular mechanism(s) of cryptdin 3-induced channel formation and down regulation. The significance of these studies is emphasized by accumulating evidence that the intestinal crypt plays a central role in intestinal epithelial cell ontogeny, physiology (solute and water transport), and specific and non-specific mucosal defense. Cryptdins act in paracrine fashion to regulate crypt cell function and appear to have a unique mechanism of action. Proposed studies will elucidate the biology of these novel alpha-defensins and may set the stage for future development of alpha-defensin inhibitors or for use of these (or related peptides) in treatment of disease at the mucosal surfaces of the intestine or respiratory tract.